Multisite regulation integrates multimodal context in sensory circuits to control persistent behavioral states in C. elegans

Maintaining or shifting between behavioral states according to context is essential for animals to implement fitness-promoting strategies. How the integration of internal state, past experience and sensory inputs orchestrates persistent multidimensional behavioral changes remains poorly understood. Here, we show that C. elegans integrates environmental temperature and food availability over different timescales to engage in persistent dwelling, scanning, global or glocal search strategies matching thermoregulatory and feeding needs. Transition between states, in each case, involves regulating multiple processes including AFD or FLP tonic sensory neurons activity, neuropeptide expression and downstream circuit responsiveness. State-specific FLP-6 or FLP-5 neuropeptide signaling acts on a distributed set of inhibitory GPCR(s) to promote scanning or glocal search, respectively, bypassing dopamine and glutamate-dependent behavioral state control. Integration of multimodal context via multisite regulation in sensory circuits might represent a conserved regulatory logic for a flexible prioritization on the valence of multiple inputs when operating persistent behavioral state transitions.


Supplementary Figure 3. Temperature and food effects on behavioral states based on motion behavioral parameters
Same analyses and displays as in Figure 1, but using only the motion parameters as defined in Supplementary Fig. 1c (a-l). Summary of the steady states after 6 h (m). Source data are provided as a Source Data file.

Supplementary Figure 4. Temperature and food effects on behavioral states based on postural behavioral parameters
Same analyses and displays as in Figure 1, but using only the postural parameters as defined in Supplementary Fig. 1c (a-l). Summary of the steady states after 6 h (m). Source data are provided as a Source Data file.

Supplementary Figure 5. Behavioral codes during food and temperature-dependent behavioral transitions
Heat-map of behavioral parameters of z-scores across the indicated conditions and hierarchical clustering based on Euclidian distance (tree on the left). Clusters of parameters affected by starvation, growth temperature and/or recent temperature shift are annotated (brackets on the right). A time series over 6 h is presented for each condition based on the same data set as the one use for PCA analyses reported in Fig. 1. Each data point represents the average value for 3-min recordings on at least three independent worm populations (≥40 worms each). Source data are provided as a Source Data file.

Supplementary Figure 6. Distribution of worms during thermotaxis assay
Histograms of on-food thermotaxis assays in wild type animals revealing a faster thermotactic movement toward recent growth temperature in scanning animals (6h after warming) as compared to dwelling animals held at 25°C as shown in Fig. 2d-f. n =10 assays each with ≥50 worms. Source data are provided as a Source Data file.

Supplementary Figure 7. Dispersal trajectory simulations comparing dwelling, global and glocal search states
Results of Monte-Carlo simulations considering the average frequency of turns and speed measured in worm populations in isothermal environments. Fifteen 1-min trajectories for each condition (a). For the sake of better visibility, dwelling trajectories are magnified (insets). Average (± s.e.m,) for animal displacement (b, corresponding to how far animals moved from their starting point) and covered distance (c, corresponding to the path length of each track). Both simulated and real data are presented side-by-side. Real data are the same as in Fig. 2b and c. **, p<.01 versus 15°C Fed condition, ##, p<.01 versus the indicated control by Bonferroni posthoc tests. ns, not significant. For simulations: data as mean ± s.e.m. of n= 200 independent simulated worm trajectories per condition. Source data are provided as a Source Data file.

Supplementary Figure 8. Dissection of global search and glocal search dispersal trajectories with controlled parameter simulations
Results of Monte-Carlo simulations considering the average frequency of turns and speed measured in worm populations in isothermal environments. Fifteen 1-min trajectories for each condition (a). Average (± s.e.m) for animal displacement (b, corresponding to how far animals moved from their starting point) and covered distance (c, corresponding to the path length of each track). Data for global search and glocal search (same as in Supplementary  Fig. 7) are presented together with controlled parameter simulation, in which only one parameter was changed at a time (either only turning rate or only speed). Data as mean ± s.e.m. of n= 200 independent simulated worm trajectories per condition (b, c). Source data are provided as a Source Data file. Figure 9. Global or glocal search states are distinct from the local search state observed 5 min after food deprivation. PCA analysis presented like in Fig. 1, showing that the Local search state after 5 min of food deprivation locates to a distinct location in the PCA space as compared to Dwelling, Scanning, Global search or Glocal search states (a). Specific behavioral states are associated with specific values for speed (b), backward frequency (c) and omega turn frequency (d). Data as mean ± s.e.m.; indicated n correspond to independent assays, each scoring ≥30 worms (b, c, d). **, p<.01 versus N2 with the same treatment by Bonferroni post-hoc tests. ns, not significant. Source data are provided as a Source Data file.

Supplementary Figure 10. Postural alteration during transition to scanning depends on AFD neuron, FLP-6 neuropeptide and multiple GPCRs
Time course of worm posture alteration (decrease in tail bending) after warming from 15 to 25°C (a). Tail bend at t=0 and t=6 h following warming (thermal shift from 15 to 25°C) in the indicated transgenic or mutant strains (b, c, d). Pgcy-8::TeTx transgene blocking synaptic transmission in AFD (b); Pgcy-8::flp-6 transgene for AFD-specific flp-6 rescue (c); Pegl-6::egl-6, transgene for egl-6 over-expression and flp-6 mutation by-pass analyses in egl-6 and flp-6 mutant background, respectively (d). # p<.05 and ##, p<.01 versus the indicated control by Bonferroni posthoc tests. ns, not significant. Control analysis with a calcium-binding impaired version of YC2.3 harboring S21A, S57A, D94S and D130A mutations showing no impact of the treatments on the YFP/CFP signal ratio in AFD (ns, not significant by two-sided Student's t-tests, e), in contrast to data with intact YC2.3 reported in Fig. 4D. On-food thermotaxis of flp-6 mutant over 3 hours, showing no significant differences (ns) between worms maintained at 25°C (25°C Fed) and worms shifted from 15 to 25 6 h prior to the assay (15-25°C Fed) (f). Data as mean ± s.e.m.; indicated n correspond to independent assays, each scoring ≥30 worms (a, b, c, d, f) or independent neurons (e). Source data are provided as a Source Data file.

Supplementary Figure 11. FLP thermosensory neurons are essential for omega turn increase during glocal search
Time course of omega turn frequency increase after starvation at 25°C (25°C Starved) as compared to controls (15°C Fed, 25°C Fed, 15°C Starved) (a). Omega turn frequency measured after 6 h of starvation at 25°C in wild type (N2), in transgenic lines with genetic ablation of the indicated neurons, or in animals carrying a Pmec-3::TeTx transgene blocking neurotransmission in FLP (b). Like for speed elevation (Fig. 5), FLP plays a major role in the up-regulation of omega turns. Control analysis with a calcium-binding impaired version of YC2.3 harboring S21A, S57A, D94S and D130A mutations showing no impact of the treatments on the YFP/CFP signal ratio in FLP (ns, not significant by two-sided Student's t-tests, c), in contrast to data with intact YC2.3 reported in Fig. 5D. Data as mean ± s.e.m.; indicated n correspond to independent assays, each scoring ≥30 worms (a, b), or independent neurons (c). Source data are provided as a Source Data file.

Supplementary Figure 14. Omega turn up-regulation during glocal search involves FLP-5/DMSR-1 signaling from FLP
Genetic dissection of the molecular signaling controlling omega turn frequency increase during glocal search. Data as mean ± s.e.m.; indicated n correspond to independent assays, each scoring ≥30 worms. Impact of neuropeptideaffecting mutations on the omega turn frequency of starved animals held at 25°C (a). Impact of flp-5 mutation, overexpression with a Pflp-5::flp-5 transgene, and rescue/over-expression with Pmec-3::flp-5 transgene expressed in FLP (b). Impact of mutations affecting FLP-5 and its receptors (c). Impact of gain-of-function (gf) and loss-of-function (lf) mutations in egl-6, as well as FLP-5 receptor over-expression in egl-6-expressing cells, revealing that EGL-6, DMSR-1a, DMSR-1b and DMSR-7 have a similar inhibitory effect on omega turn frequency in starved animals at 25°C (d). No effect of DMSR-1 overexpression in dmsr-1-expressing cells or AVA-specific overexpression, respectively, on omega turn frequency in starved animals at 25°C (e). **, p<.01 versus N2; ##, p<.01 versus the indicated condition by Bonferroni posthoc tests. ns, not significant. Source data are provided as a Source Data file.